The goal of this project is to optimize potent and selective peptide inhibitors of human voltage-gated sodium (Nav) channels for treatment of chronic pain. Two recent breakthroughs: (1) high resolution structures revealing human Nav channel interactions with peptide toxins, and (2) Rosetta computational approaches for in silico optimization of high affinity, specificity and stability of peptide mimics, have together set the stage for radical optimization of biologics as superior therapeutic candidates for the treatment of chronic pain. Pain signals originate predominantly in a subset of peripheral neurons that harbor a distinct subset of Nav channels. Clinical use of Nav channel blockers, such as local anesthetics, is widespread, as they are non-addictive and can provide complete pain relief by inhibiting Nav channels in peripheral sensory neurons. However, local anesthetics are non-selective and also block Nav channels vital for function of the heart, muscle, and central nervous system. One of the key challenges for the development of novel analgesics is selectively targeting Nav channel subtypes involved in pain signaling. Genetic studies have identified human Nav1.7, Nav1.8, and Nav1.9 channel subtypes as key players in pain signaling. These Nav channels are major contributors to action potential generation in peripheral neurons and are established molecular targets for pain therapy. Nav channels are also prime targets of peptides from venoms of predatory creatures. ProTx-II is a highly potent and moderately selective peptide toxin that inhibits human Nav1.7 activation. Recent high-resolution structures of ProTx-II ? human Nav1.7 channel complexes have identified structural regions forming the toxin-channel interface and revealed the molecular mechanism of toxin action. Importantly, this new atomistic structural data allows application of recent breakthroughs in Rosetta computational peptide design approaches. We propose radical optimizations of ProTx- II selectivity, potency, and stability by exploiting the new structures of ProTx-II ? human Nav1.7 channel complexes, advances in rational peptide optimization, and rigorous potency and efficacy screens to generate high-affinity, selective inhibitors of human Nav1.7, Nav1.8, and Nav1.9 channels. Novel, high-affinity, selective inhibitors of these Nav channels have transformative potential to define a new class of biologics to treat pain.